Media Recipes used in CMAR 

ASM (see MLA)

BB

BG

D

DYIY

F, F/2, FE, FE/2

GP     Gse

JM

K

MBL

MJ

MLA

MMII

Porphyridium

Soil extract

Media Comparison Table (constituents in μmoles of some of the above media)

Further help on making media including characteristics of individual media components

See notes for Aquaculturists if large volumes of media are needed

Seawater source

 

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Bolds Basal Medium

References:

Nichols, H. W. and Bold, H. C. (1965). Trichosarcina polymorpha gen. Et sp. Nov. J. Phycol. 1: 34-8.

Nichols, H. W. (1973) Growth media – freshwater. In Stein, J. (Ed.) Handbook of Phycological Methods, Culture Methods and Growth Measurements, Camb. Univ. Press pp. 7-24.

 

Adapted for freshwater algae

 

Stock Solutions	per Litre distilled water (dH2O)
1. NaNO3	25.0 g
2. CaCl2.2H2O	2.5 g
3. MgSO4.7H2O	7.5 g
4. K2HPO4	7.5 g
5. KH2PO4	17.5 g
6. NaCl	2.5 g
7. EDTA      KOH	50.0 g 31.0g
8. FeSO4.7H2O       H2SO4	4.98 g 1.0 mL
9. H3BO3	11.42 g
10. Micronutrients	g.L-1	Add each constituent separately to ~800 mL of dH2O and fully dissolve between each addition.  Then make up to 1L.
ZnSO4.7H2O	8.82 g
MnCl2.4H2O	1.44 g
MoO3	0.71 g
CuSO4.5H2O	1.57 g
Co(NO3)2.6H2O	0.49 g

 

Store all stock solutions in the refrigerator.

 

To Prepare BB Medium – standard method

Add 10 mL of each stock solution (1 – 6) to 940 mL distilled water.

Add 1 mL of stock solutions (7 – 10).

Autoclave at 121°C (15PSI for 15 mins).

 

To Prepare BB Medium – method combining stocks from different media used in CMAR

 

1. NaNO3                                                                          2.5 mL D stock solution

2. CaCl2.2H2O                                     0.5 mL D stock solution

3. MgSO4.7H2O                                   0.94 mL MMII stock solution

4. K2HPO4                                                                        2.5 mL D stock solution

5. KH2PO4                                                                        10.0 mL BB stock solution

6. NaCl                                                10.0 mL BB stock solution

7. Na2EDTA                                        1.0 mL of 57.05gL-¹H2O stock

8. FeSO4.7H2O with H2SO4                 1.0 mL BB stock solution

9. H3BO3                                                                            1.0 mL BB stock solution

10. Micronutrients                               1.0 mL BB stock solution

 

Add each stock solution (1 – 10) in the stated volume to 1 litre distilled water.

Autoclave at 121°C (15PSI for 15 mins).

 

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BG (Blue - Green Medium)

Medium for Spirulina spp.

Source: Culture Collection of Algae and Protozoa Catalogue of Strains (1988).

 

Stock Solutions	per litre distilled water (dH2O)
1. NaNO3	15.0 g
2. K2HPO4.3H2O	4.0 g
3. MgSO4.7H2O	7.5 g
4. CaCl2.2H2O	3.6 g
5. Citric acid	0.6 g
6. Ferric ammonium citrate	0.6 g (Autoclave to dissolve)
7. EDTA	0.1 g
8. Na2CO3	2.0 g
9. Trace metal mixture	g.L-1	Add each constituent separately to ~800 mL of dH2O and fully dissolve between each addition.  Then make up to 1 L.
H3BO3	2.86 g
MnCl2.4H2O	1.81 g
ZnSO4.7H2O	0.222 g
Na2MoO4.2H2O	0.39 g
CuSO4.5H2O	0.079 g
Co(NO3)2.6H2O	0.0494 g

 

To Prepare 1 litre of media

To 829 ml distilled water add:

Stock solution 1           100 ml

Stock solution 2-8       10 ml each

Stock solution 9           1 ml

 

pH should be adjusted to approximately 7.4 with 1M NaOH or HCl before autoclaving.

 

BG Medium with Seawater - CSIRO modification

To prepare Medium

Step 1

Seawater mix

To 971 mL of Teflon sterilised seawater add the following:

4.0 ml per litre of G vitamins - filtered sterilised.

25 ml per litre sterile soil extract.

Step 2

Final BG/SW medium

 

Mix aseptically equal parts of Step 1 Seawater mix with BG medium recipe.

 

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D MEDIUM

 

Distilled H2O	1.0 liter
NaCl	18.0 g
MgSO4, 7H2O	5.0 g
KCl	6.0 mL of 10% solution
NaNO3	5.0 mL of 10% solution
CaCl2.2H2O	2.77 mL of 10% solution
K2HPO4	0.3 mL of 10% solution
FeCl3.6H20	0.05 mL of 10% solution
TRIS	10.0 mL of 10% solution
B12	3.0 mL of 1µg/mL solution
Thiamine	1.0 mL of 1mg/mL solution
P II Metal Mix	5.0 mL of stock solution (see below)

Adjust pH to 7.6-7.8 with 1 N HCl (approx.5-6 mls/liter)

 

 

P II Metal Mix Stock Solution	g / L	Add the Na2EDTA to ~750mL of dH2O in a volumetric flask and stir over low heat to dissolve.  Add each of the other constituents separately to ~200mL of dH2O and fully dissolve between additions.  Then add this second solution to the Na2EDTA and make up to 1 L.
Na2EDTA	6.0 g
FeCl3.6H2O	0.29 g
H3BO3	6.85 g
MnCl2.4H2O	0.86 g
ZnCl2	0.06 g
CoCl2.6H2O	0.026 g
(adjust pH to 7.8 - 8.0 with NaOH)

 

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DYIY Medium (freshwater)

References

(Keller and Anderson unpubl.; modified from Lehman 1976)

Source: In: Journal of Phycology, 1997, supplement to Vol. 33, No. 6, CCMP - Provasoli-Guillard National Center for Culture of Marine Phytoplankton

 

Stock Solutions	Per 500mL distilled water (dH2O)
1. MgSO4 .7H2O	25.0 g
2. KCl	1.5 g
3. NH4NO3	1.33 g
4. NaNO3	2.5 g
5. Na2 glyceroPO4	5.0 g
6. H3BO3	0.4 g
7. Na2EDTA	4.0 g
8. NaSiO3. 9H2O	7.5 g
9. FeCl3 .6H2O	1.35 g
10. CaCl2 .2H2O	37.5 g
11. f/2 vitamins (see below)	1 mL
12. trace metals (see below)	1 mL

 

f/2 vitamin Stock Solution
Working Stock
to one liter of distilled water, add the following:
Biotin	10.0 mL primary stock
Vitamin B12	1.0 mL primary stock
Thiamine HCL	200.0 mg
Primary Stocks
Biotin	0.1 mg. mL-
Vitamin B12	1.0 mg. mL-1

 

DY trace metals mg per 500 mL distilled water
MnCl2. 4H2O	100 mg	Add each constituent to ~400ml dH2O, fully dissolving between additions. Then make up to 500ml with dH2O
ZnSO4.7H2O	20 mg
Na2MoO4 . 2H2O	10 mg
CoCl2 . 6H2O	4 mg
H2SeO3	2 mg
Na3VO4 .nH2O	1 mg

 

To prepare 1L of medium

1.  To 900 mL of dH2O add 200 mg of MES buffer

2.  Add 1 mL of the stock solutions (1-12).

3.  After all additions, bring to 1 L and adjust the pH to 6.8 (it will probably be very acidic).

 

DYIY (freshwater) Medium - CSIRO Modification

References (Keller and Anderson unpubl.; modified from Lehman 1976)

Source: In: Journal of Phycology, 1997, supplement to Vol. 33, No. 6, CCMP - Provasoli-Guillard National Center for Culture of Marine Phytoplankton

 

To 900 ml of ddH2O add 200 mg of MES buffer and dissolve. Add the following stock solutions. After all additions, make up to 1 L. Adjust the pH to 6.8. Autoclave.

 

Stock Solution                                                            Source

MES                                        200 mg / L

MgSO4.7H2O                           24.7 g / 500 mL                            MLA stock use 1.0 mL

KCl                                         1.5 g / 500 mL                               DYIY stock use 1.0 mL

NH4NO3                                                         1.33 g / 500 mL                            DYIY stock use 1.0 mL

NaNO3                                                             2.5 g / 500 mL                                 DYIY stock use 1.0 mL

Na2 glyceroPO4                                       1.62 g / 500 mL                            K stock use 3.0 mL

H3BO3                                                              1.24 g / 500 mL                            MLA stock use 0.3 mL

Na2EDTA                                2.18 g / 500 mL                            MLA stock use 1.8 mL

NaSiO3.5H2O                           11.35 g / 500 mL                       f stock use 0.66 mL

FeCl3.6H2O                              0.79 g / 500 mL                            MLA stock use 1.7 mL

CaCl2.2H2O                             14.7 g / 500 mL                            MLA stock use 2.6 mL

f/2 vitamins (see below) use 1.0 mL

trace metals (see below) use 1.0 mL

 

f/2 vitamin Stock Solution

Working Stock

to 100ml of distilled water, add the following:

Biotin                                      1.0 mL primary stock

Vitamin B12                            1.0 mL primary stock

Thiamine HCL                                     200.0 mg

            Primary Stocks

            Vitamin B12                0.1 mg / mL

            Biotin                          0.1 mg / mL

 

DY trace metals

Add to 500 mL distilled water, first dissolving seperately:

MnCl2.4H2O                            100 mg

ZnSO4.7H2O                              20 mg

Na2MoO4.2H2O                         10 mg

CoCl2.6H2O                                 4 mg

Na3VO4 .nH2O                 1 mg

Add as the following stock solution:

H2SeO3                                                            0.645 mg GSe stock use 3mL

 

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Medium f (and fE) - CSIRO Modification

This medium is a slight modification of the original f medium of Guillard and Ryther (1962).  It is most commonly prepared at half strength where it is designated as f2 or f/2.

 

Reference: Jeffrey, S. W. and LeRoi, J.-M. (1997). Simple procedures for growing SCOR reference microalgal cultures. In: S.W. Jeffrey, R.F.C. Mantoura and S.W. Wright (Eds) Phytoplankton pigments in oceanography; Monographs on oceanographic methodology 10, UNESCO, France, pp 181-205.

 

Guillard, R. R. L. and Ryther, J. H. (1962) Canad. J. Microbiol., 8: 229-239.

 

Stock Solutions	Per L distilled water (dH2O)
1. NaNO3	150.0 g
2. Trace metals	mg / L	Add each of the constituents to ~750ml dH2O, mixing thoroughly between additions to dissolve.  Finally make solution up to 1L
CuSO4.5H2O	19.6 mg
ZnSO4.7H2O	44.0 mg
CoCl2.6H2O	22.0 mg
MnCl2.4H2O	360.0 mg
Na2MoO4.2H2O	12.6 mg
3. Na2SiO3.5H2O	22.7 g
4. Fe citrate:	g / L	add both constituents to 1L of dH2O and autoclave to dissolve
Ferric citrate	9.0 g
Citric acid	9.0 g
5. Vitamins
(i)Working Stock Solution (make fresh solution every 3 months)	to 100 mL of distilled water, add the following
Biotin	1.0 mL primary stock
Vitamin B12	1.0 mL primary stock
Thiamine HCL	20.0 mg
(ii) Primary Stocks
Vitamin B12	10.0 mg /100 mL dH2O
Biotin	10.0 mg /100 mL dH2O
6. NaH2PO4.2H2O	11.3 g
7. Na2EDTA.2H2O	30.0 g

Store all stock solutions in the refrigerator.

 

Preparation Methods

1. To Prepare Medium f

Add 1 mL of each stock solution (1 – 5) to 1 litre seawater. Dispense to flasks and autoclave at 121°C (15PSI, 15 mins).

 

Phosphate (see Stock 6.- NaH₂PO₄.2H₂O ). This must be sterilised separately from seawater to prevent precipitation.  Dilute original phosphate stock with distilled water such that 1 mL added to each flask of sterile medium will give the required concentration of phosphate in the medium. Autoclave dilute phosphate stock at 121°C (15PSI, 15 mins). After cooling, dispense aseptically with sterilised automatic dispenser.

 

For example:

For 100 x 125 mL Erlenmeyer flasks, each containing 75 mL medium, prepare dilute phosphate stock as follows:

f and fE media:

Take 7.5 mL of original phosphate stock and make up to 100 mL with distilled water.

Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL per flask aseptically.

 

f2 and fE2 media:

Take 3.75 mL of original phosphate stock and make up to 100 mL with distilled water.

Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL per flask asepically.

 

Scale up in the same proportion for larger volumes.

 

To Prepare Medium fE

Prepare as medium f, but also add 1 mL of Na₂EDTA.2H₂O stock solution (7).

 

To Prepare Medium f2

Prepare as medium f, but using 0.5 mL of each stock solution instead of 1.0 mL of each.

 

To Prepare Medium fE2

Prepare as medium f2, but also add 0.5mL of Na2EDTA.2H2O stock solution.

 

2. To Prepare Medium f2 concentrated nutrients

Take 5mL of each stock solution (1 – 6) and make up to 100 mL with distilled water.

Pour into a 250 mL Schott bottle.

Autoclave at 121°C (15PSI, 15 mins).

Alternatively, filter sterilise using a 0.22 mm filter into a sterile 250 mL Schott bottle.

 

Use 1 mL per 100 mL sterile seawater adding correct amount of nutrient aseptically.

 

See notes for Aquaculturists if a greater volume of concentrated nutrients is needed

 

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G. P. medium (CMAR uses the abbreviation G for GP medium)

Reference: Loeblich, A. R. and Smith, V. E. (1968) Lipids, 3: 5-13.

 

Note about Salinity: In medium G and derivatives the final preparation steps require mixing of nutrients with seawater and distilled water in a 3:1 or 4:1 ratio.  The fully marine seawater (~33-36practical salinity units) used in CMAR means the resulting media salinity is ~28psu.  In our culturing experience this is a good salinity for estuarine and coastal flagellate species, particularly dinoflagellates.

 

Stock Solutions	Per Litre distilled water (dH2O)
1. KNO3	100.0 g
2. K2HPO4	34.8 g
3. Vitamins
Working Stock Solution	to 100 mL of distilled water, add the following
Biotin	2.0 mL primary stock
Vitamin B12	1.0 mL primary stock
Thiamine HCL	100.0 mg (fresh solution every 3 months)
Primary Stocks
Vitamin B12	10.0 mg /100 mL dH2O
Biotin	10.0 mg /100 mL dH2O
4. PII Metal Mix	g / L	Add the Na2EDTA to ~750mL of dH2O in a volumetric flask and stir over low heat to dissolve.  Add each of the other constituents separately to ~200mL of dH2O and fully dissolve between additions.  Then add this second solution to the Na2EDTA and make up to 1L.
Na2EDTA	6.0 g
FeCl3.6H2O	0.29 g
H3BO3	6.85 g
MnCl2.4H2O	0.86 g
ZnCl2	0.06 g
CoCl2.6H2O	0.026 g
(adjust pH to 7.8 - 8.0 with NaOH)
5. Soil Extract see recipe at end

Store all stock solutions in the refrigerator.

 

G. P. medium

Preparation Methods

1. To Prepare G medium

To 750 mL seawater add:

Distilled water              250 mL

Nitrate stock                     2 mL

Vitamin stock                   1 mL

PII Metal Mix                   5 mL

Soil Extract                      5 mL

Dispense to flasks and autoclave at 121°C (15PSI, 15 mins).

 

Phosphate must be sterilised separately from seawater to prevent precipitation.

Dilute original phosphate stock with distilled water such that 1 mL added to each flask of sterile medium will give the required concentration of phosphate in the medium. Autoclave dilute phosphate stock at 121°C (15PSI, 15 mins). After cooling, dispense aseptically with sterilised automatic dispenser.

For example:

For 100 x 125 mL Erlenmeyer flasks, each containing 75 mL medium, prepare dilute phosphate stock as follows:

G medium:

Take 7.5 mL of original phosphate stock and make up to 100 mL with distilled water.

Pour into a 250 mL Schott bottle and autoclave to sterilse. Dispense 1 mL per flask aseptically.

G₂ medium:

Take 3.75 mL of original phosphate stock and make up to 100 mL with distilled water.

Pour into a 250 mL Schott bottle and autoclave to sterilize. Dispense 1 mL per flask aseptically.

Scale up in the same proportion for larger volumes.

 

To Prepare Medium G₂

Make up dilutant, consisting of seawater and distilled water (3:1 ratio)

Dilute G medium by adding an appropriate volume of dilutant such that:

- medium is half of its original concentration (G₂ medium).

 

2. To Prepare Medium G concentrated nutrients

Add Nitrate stock                    20 mL

Vitamin stock                          10 mL

PII Metal Mix                          50 mL

Soil Extract                             50 mL

Phosphate stock                       10 mL

 

Make up to 200 mL with distilled water.

Pour into a 250 mL Schott bottle.

Autoclave at 121°C (15PSI, 15 mins).

 

Alternatively, filter sterilise using a 0.22 mm filter into a sterile 250 mL Schott bottle.

Use 2mL /100mL sterile seawater and distilled water (3:1 ratio).

Add correct amount of nutrients aseptically.

 

To Prepare Medium G2 concentrated nutrients

Use G concentrated nutrients such that:

G2: use 1 mL /100mL sterile seawater and distilled water (3:1 ratio).

G5: use 0.4 mL /100mL sterile seawater and distilled water (3:1 ratio).

 

GSe medium – Modification of G. P. medium

Reference: Blackburn, S. I.; Bolch, C. J. S.; Haskard, K. A., and Hallegraeff, G. M. Reproductive Compatibility Among Four Global Populations of the Toxic Dinoflagellate Gymnodinium Catenatum (Dinophyceae). Phycologia. 2001; 40(1):78-87.

Medium GSe –selenium is added as an important trace metal (chelator).

 

Note about Salinity: In medium G and derivatives the final preparation steps require mixing of nutrients with seawater and distilled water in a 3:1 or 4:1 ratio.  The fully marine seawater (~33-36 practical salinity units) used in CMAR means the resulting media salinity is ~28 psu.  In our culturing experience this is an optimal salinity for estuarine and coastal flagellate species, particularly dinoflagellates.

 

Stock Solutions	Per Litre distilled water (dH2O)
1. KNO3	100.0 g
2. K2HPO4	34.8 g
3. Vitamins
Working Stock Solution	to 100 mL of distilled water, add the following
Biotin	2.0 mL primary stock
Vitamin B12	1.0 mL primary stock
Thiamine HCL	100.0 mg (fresh solution every 3 months)
Primary Stocks
Vitamin B12	10.0 mg /100 mL dH2O
Biotin	10.0 mg / 100 mL dH2O
4. PII Metal Mix	g / L	Add the Na2EDTA to ~750 mL of dH2O in a volumetric flask and stir over low heat to dissolve.  Add each of the other constituents separately to ~200 mL of dH2O and fully dissolve between additions.  Then add this second solution to the Na2EDTA and make up to 1L.
Na2EDTA	6.0 g
FeCl3.6H2O	0.29 g
H3BO3	6.85 g
MnCl2.4H2O	0.86 g
ZnCl2	0.06 g
CoCl2.6H2O	0.026 g
(adjust pH to 7.8 - 8.0 with NaOH)
5. Soil Extract	See soil extract protocol
6. Selenium (as selenite) H2SeO3	1.29 mg

Store all stock solutions in the refrigerator.

 

GSe medium

Preparation Method

1. Seawater

Autoclave filtered seawater in 1000 mL Teflon bottles to sterilise.

 

2. Distilled Water

Autoclave distilled water to sterilise.

 

3. To Prepare GSe medium concentrated nutrients (excluding soil extract)

Add Nitrate stock                    20 mL

Phosphate stock                       10 mL

Vitamin stock                          10 mL

PII Metal Mix                          50 mL

Selenium stock                        10 mL

 

Make up to 200 mL with distilled water.

Pour into a 250 mL Schott bottle.

Autoclave at 121°C (15PSI, 15 mins).

 

Alternatively, filter sterilise using a 0.22 mm filter into a sterile 250 mL Schott bottle.

Use 2 mL /100mL sterile seawater and distilled water (3:1 ratio).

Add correct amount of nutrients aseptically.

 

4. Soil Extract Solution

See soil extract protocol for details.

Use soil extract at a concentration of 0.5 mL per 100 mL medium.

 

To Prepare GSe medium

For example to make 5000 mL Medium GSe:

In a sterile 5000 mL Schott bottle add aseptically

 

sterile seawater (1)                                         4000 mL

sterile distilled water (2)                                1000 mL

sterile GSe concentrated nutrients (3)                           100 mL

sterile soil extract solution (4)                                        25 mL

Mix. This medium is now ready to be decanted aseptically into sterile culture flasks.

 

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Soil Extract

Soil Extract is an adaptation of E.G. Pringsheim's biphasic soil-water medium and is a component of some of our media. As the chemical composition is not well-defined and may vary from batch to batch its use in experimental situations is not recommended, however in our experience certain species will not grow well without it and soil extract is added for both culture maintenance and experimental studies.  An extensive list of modified Soil Water media developed for a range of microalgae from specific environments can be found on the UTEX website (The Culture Collection of Algae at the University of Texas).

http://www.utex.org/

 

CMAR protocol

Soil must be collected from a natural uncultivated environment or a rich garden loam may be suitable. No fungicides, insecticides, garden fertilizers or fresh manure should be present. At CSIRO, topsoil from a local sandy bushland environment has proved to have particularly beneficial growth promoting properties. Soil from clay or other soil types are less suitable in our experience.

Soil should not be stored or processed in the algal culture laboratory, since it is a potent source of unwanted microorganisms. Soil should be aged under moist conditions (preferably for 6 months or more) and then kept dry and away from light.

 

To Prepare Soil Extract

1. Sift dry soil (not recently treated with fertilizer or herbicide) once through a coarse sieve and twice through a fine sieve (1mm mesh).

2. Mix 1 kg of soil into 2 litres of distilled water.

3. Autoclave for 60 minutes at 121°C and cool overnight.

4. Filter through absorbant cotton wool packed into the stem of a glass filter funnel.

5. Centrifuge at 5000 rpm for 20 minutes in 250 ml polyethylene centrifuge tubes and collect the deep brown supernatant.

6. Filter again through absorbant cotton wool.

7. Dispense the supernatant (50 mL aliquots) into 100 mL Schott bottles or 100 mL media bottles.

8. Autoclave for 15 minutes at 121°C.

9. After cooling, wrap caps with parafilm to prevent airborne contamination from fungal spores or bacteria.

10. Store sterile soil extract at 4°C (cold room or refrigerator).

 

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JM (Jaworski’s Medium)

Reference: Culture Collection of Algae and Protozoa (CCAP) – Catalogue of Strains 1988.

Adapted for freshwater Algae

 

Stock Solutions	Per Litre distilled water (dH2O)
1. Ca(NO3)2.4H2O	20.0 g
2. KH2PO4	12.4 g
3. MgSO4.7H2O	50.0 g
4. NaHCO3	15.9 g
5. EDTA FeNa	2.25 g
EDTA Na2	2.25 g
6. H3BO3	2.48 g
MnCl2.4H2O	1.39 g
(NH4)6MO7O24.4H2O	1.00 g
7. Vitamins
Cyanocobalamin (Vitamin B12)	0.04 g
Thiamine HCl (Vitamin B1)	0.04 g
Biotin	0.04 g
8. NaNO3	80.0 g
9. Na4HPO4.12H2O	36.0 g

Store all stock solutions in the refrigerator.

 

To Prepare JM - fully autoclaved media for axenic cultures

Add 1 mL of each stock solution (1 – 9) to 1 litre distilled water.

Autoclave at 121°C (15 PSI for 15 mins).

 

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K Medium

Reference: Adapted from Table 2; Keller, M. D., Selvin, R. C., Claus, W. and Guillard, R. R. L. (1987).  Media for the culture of oceanic ultraplankton. J. Phycol. 23, 633-638

 

Note: Specific K stock solutions and stock solutions from other media are used in CMAR to prepare this media.  A concentrated working stock solution is made up and used at a concentration of 2mL per 100ml of seawater..

 

To prepare 100 ml working stock : (use at 2 mL per 100 mL seawater)

 

Stock Solutions          primary stock             source             Volume

1. NaNO₃                     150 g / L H₂O              f stock solution, use                 2.5 mL

2. Na₂glyceroPO₄        3.24 g / L H₂O             K stock solution, use               5 mL

3. Na₂SiO3.9H2O         22.7 g / L H₂O             f stock solution, use                 2.5 mL

4. Vitamins                                                      f stock solution, use                 5 mL

5. Trace Metals

       FeNaEDTA          4.3 g / L H₂O               K stock solution, use               5 mL

       K-Primary Trace mix (see below)           K stock solution, use               5 mL

       Na₂EDTA.2H2O 30 g / L H₂O                  fE stock solution, use              6.2 mL

           

6. TRIS                        100 g / L H₂O              K stock solution, use               6.05 mL

7. NH₄Cl                     2.68 g / L H₂O             K stock solution, use               5.0 mL

8. H₂SeO₃                    1.29 mg / L H₂O          GSe stock solution, use           5.0 mL

9. Distilled water                                                                                            52.75 mL

           

Filter-sterilise through 0.22 µm sterile filter under aseptic conditions.

 

K-Primary Trace mix

CuSO₄.5H₂O                  4.9 mg / L H₂O

ZnSO₄.7H₂O                22.0 mg / L H₂O

CoCl₂.6H₂O                 11.0 mg / L H₂O

MnCl₂.4H₂O                180.0 mg / L H₂O

Na₂MoO₄.2H₂O              6.3 mg / L H₂O

Add each ingredient to ~750 mL of distilled water, mixing

thouroughly between additions and then make up to 1 L.

 

All stock solutions are made up in distilled water.

Store all stock solutions in the refrigerator.

 

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MBL Medium - Woods Hole

Reference: Nichols, H. W. (1973) in Handbook of Phycological Methods, Ed. J. R. Stein, pp. 16-17. Camb. Univ. Press. (R. R. L. Guillard, personal communication).

 

Adapted for freshwater Algae

 

Stock solutions	Per Litre distilled water (dH2O)
1. CaCl2.2H2O	36.76 g
2. MgSO4.7H2O	36.97 g
3. NaHCO3	12.60 g
4. K2HPO4	8.71 g
5. NaNO3	85.01 g
6. Na2SiO3.9H2O	28.42 g
7. Na2EDTA	4.36 g
8. FeCl3.6H2O	3.15 g
9. Metal Mix		Add each constituent separately to ~750mL of dH2O, fully dissolving between aditions.  Finally make up to 1L with dH2O.
CuSO4.5H2O	0.01 g
ZnSO4.7H2O	0.022 g
CoCl2.6H2O	0.01 g
MnCl2.4H2O	0.18 g
Na2MoO4.2H2O	0.006 g
10. Vitamin stock
Cyanocobalamin (Vitamin B12)	0.0005 g / L dH2O
Thiamine HCl (Vitamin B1)	0.10 g / L dH2O
Biotin	0.0005 g / L dH2O
11. Tris stock	250.0 g / L dH2O

Store all stock solutions in the refrigerator.

 

 

To Prepare MBL Medium

Add 1mL of each stock solution (1 – 11) to 1 litre distilled water.

(For species which cannot use nitrate substitute 1mL of NH₄Cl made up to 5.4 g /L H2O)

Adjust pH to 7.2 with HCl.

Autoclave at 121°C (15PSI for 15 mins).

 

To Prepare MBL/NB2 Medium

Make MBL medium as above and add 2.5 g Oxiod nutrient broth No. 2 before autoclaving.

 

MBL Medium - Woods Hole: CSIRO working modification

This recipe closely resembles the Woods Hole MBL Medium listed previously. However it is made up using stocks from other media used in CMAR.

 

Stock Solution                        Source                        Add V

1. CaCl2.2H2O                         D stock solution          0.8 mL

2. MgSO4.7H2O                       MMII stock solution    0.5 mL

3. NaHCO3                                                  12.60 g / L-¹H2O          1.0 mL

4. K2HPO4                                                    G stock solution          0.2 mL

5. NaNO3                                                      f stock solution            0.5 mL

6. Na2SiO3.5H2O                     f stock solution            4.5 mL

7. Na2EDTA                            fE stock solution          0.15 mL

8.         Fe citrate:

Ferric citrate

Citric acid                   f stock solution            1.0 mL

9. Trace Metals                       f stock solution            0.5 mL

10. Vitamin stock                    f stock solution            1.0 mL

11. Tris stock                          D stock solution          2.0 mL

 

Store all stock solutions in the refrigerator.

For species which cannot use nitrate substitute 1mL of NH₄Cl made up to 5.4 g / L H2O

Adjust pH to 7.2 with HCl.

 

 

To Prepare MBL Medium

Add each stock solution (1 – 11) in the volumes indicated to 1 litre distilled water.

Autoclave at 121°C (15PSI for 15 mins).

 

To Prepare MBL/NB2 Medium

Make MBL Medium as above and add 2.5g Oxiod nutrient broth No. 2 before autoclaving.

 

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MJ Medium (Modified Jorgensen’s media for diatoms)

Based on recipe obtained from University of Tasmania – Harmful Algae Culture Collection

            This media has been used predominantly for the culture of Pseudonitzschia species and also in trials of some Chaetoceros species where biomass and maintenance of normal morphology seems to be better than in other media.  Preparation of the media is based on the utilization of MJ and f-stock solutions.

 

Stock Solution                        Source                                                Add V (mL.L^-1)

                                                                                                                        (a) Normal           (b) concentrated

1. NaNO3                                  f stock solution (150.0 g / L )              2.0 mL             20.0

2. K2HPO3                                MJ stock solution

                                                (make up at 2.0 g / L )                         5.0 mL             50

3. Trace Metals I         MJ stock solution (see below)                        0.1 mL             1.0

4. Trace Metals II        MJ stock solution (see below)                        1.0 mL             10.0

5. Vitamin B12                                        f stock solution (1.0 mg / L )               0.5 mL             5.0

6. Na2SiO3.5H2O         f stock solution (22.7 g / L )                4.5 mL             45

7. EDTA                      MJ stock solution (10 g / L )               1.0 mL             10

 

Autoclave each stock solution except the Vitamin B12 which should be 0.2um filter sterilised.

 

(a)    lists the normal stock volumes needed to prepare 1L of media. (b) lists the volumes to prepare a concentrated nutrient solution as described under Preparation below.

 

3. Trace Metals I

Na2EDTA	2.5 g / L H2O	Add the Na2EDTA to ~750mL of dH2O in a volumetric flask and stir over low heat to dissolve.  Add each of the other constituents separately to ~200 mL of dH2O and fully dissolve between additions.  Then add this second solution to the Na2EDTA and make up to 1 L.
CoCl2.6H2O	150 mg / L  H2O
CuSO4.5H2O	800 mg / L  H2O
(NH4)6Mo7O24	150 mg / L  H2O
NH4VO3	250 mg / L  H2O
ZnSO4.7H2O	250 mg / L  H2O

 

4. Trace Metals II

FeSO4.7H2O	3.0 g / L	Make up each constituent separately in ~200 mL of dH2O and fully dissolve.  Then combine each solution and make up to 1 L.
Citric acid	3.0 g / L
H3Bo3	1.5 g / L
MnCl2.4H2O	1.0 g / L

 

To Prepare concentrated nutrient stock (enough for 10L media)

Add aseptically each of the prepared sterile stock solutions to 59 mL of sterile distilled water in a 200 mL Schott bottle (making total volume 200 mL). 

 

To make media, add 20 ml of this final nutrient solution per 1itre of seawater.

 

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MLA Medium

Reference: Bolch, C. J. S. and Blackburn S. I. (1996). Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. Journal of Applied Phycology 8, 5-13

MLA is derived from ASM-1 medium reported in Gorham etal, (1964). Isolation and culture of toxic strains of Anabaena flos-aquae (Lyngb.) de Bréb. Verh. int. Ver. Limnol 15, 796-804

For Cyanobacterial Cultures

 

Stock Solutions	Per Litre distilled water (dH2O)
1. MgSO4.7H2O	49.4 g
2. NaNO3	85.0 g
3. K2HPO4	6.96 g
4. H3BO3	2.47 g
5. H2SeO3	1.29 mg
6. Vitamins
Working Stock Solution
to 100mL of distilled water, add the following:
Biotin	0.05 mL primary stock
Vitamin B12	0.05 mL primary stock
Thiamine HCl	10.0 mg
Primary Stocks
Biotin	10.0 mg / 100 mLH2O
Vitamin B12	10.0 mg / 100 mLH2O
7. Micronutrients
Stock Solution	.
to 800mL of distilled water add each of the following constituents separately, mixing to dissolve each addition
Na2EDTA	4.36 g (add first & stir on low heat to                fully dissolve)
FeCl3.6H2O	1.58 g
NaHCO3	0.60 g
MnCl2.4H2O	0.36 g
then add 10mL of the following primary stocks (each made up separately)
Primary Stocks	(per Litre dH20)
CuSO4.5H2O	1.0 g.
ZnSO4.7H2O	2.2 g.
CoCl2.6H2O	1.0 g.
Na2MoO4.2H2O	0.6 g.
Finally, make up the micronutrient stock to 1 litre with distilled water If precipitate forms increase pH up to 7. (If precipitation becomes an issue then replacing the two sulphate stocks with equimolar amounts of the trace metal in the chloride form has proven useful; Ben Long, pers comm)
8. NaHCO3	16.9 g
9. CaCl2.2H2O	29.4 g

Store all stock solutions in the refrigerator.

 

MLA Medium Preparation Methods

 

There are 4 components as follows:

 

1. Distilled Water

Autoclave to sterilise

2. To Prepare MLA Medium x40 concentrated nutrients (250mL volume)

To 130mL distilled water add

MgSO4.7H2O                           10 mL

NaNO3                                                             20 mL

K2HPO4                                                          50 mL

H3BO3                                                              10 mL

H2SeO3                                                            10 mL

Vitamin stock                          10 mL

Micronutrient stock                 10 mL

Filter sterilise using a 0.22 mm filter into a sterile 250 mL Schott bottle.

 

3. NaHCO3 16.9 g / L H2O

Autoclave to sterilise.

4. CaCl2.2H2O 29.4 g / L H2O

Autoclave to sterilse.

 

To Prepare MLA Medium

For example to make 1000 mL MLA Medium:

In a sterile 1000 mL Schott bottle add aseptically

sterile distilled water (1)                                964 mL

sterile MLA x40 concentrated nutrients (2)             25 mL

sterile NaHCO3 (3)                                          10 mL

sterile CaCl2.2H2O (4)                                                1 mL

Mix well after each addition.

This medium is now ready to be decanted aseptically into sterile culture flasks.

 

To Prepare MLA Medium (Fully Autoclaved)

For axenic cultures. The media is essentially the same but due to the autoclaving process the NaHCO3 concentration is adjusted.

 

For example to make 1000 mL autoclaved MLA Medium add

distilled water (1)                                           973 mL

sterile MLA x40 concentrated nutrients (2)            25 mL

sterile NaHCO3 (3)                                                                     1 mL

sterile CaCl2.2H2O (4)                                                1 mL

 

Adjust pH to 7.5 to 8.0 with HCl (often no adjustment is necessary).

Dispense to flasks and autoclave at 121°C (15PSI, 15 mins).

Allow to cool in autoclave overnight. Helps to minimise the amount of precipitate.

 

 

Protocols for solid MLA medium using a X40 concentrate

MLA for solid media is modified by the addition of Na₂SO₃, as this can improve survival of cyanobacteria on solid media (Parker, 1982). A stock solution of Na₂SO₃ (12.6 g/L) is sterilised by autoclaving. This is then added aseptically just prior to pouring to give a final concentration 0.1 mM Na₂SO₃ (i.e. 1 ml / L of Na₂SO₃stock).

 

·          Agar - final concentration of 10 g / L (1%)

- preferred media for Nodularia, but always use highly purified agar e.g. Difco Bacto purified agar.

 

·          Agarose - Final concentration of 5 g / L (0.5%)

- preferred media for Microcystis and Anabaena. (although the latter grows only very slowly, or not at all on solid media).

            · To prepare 500 ml of non-saline solid MLA medium:
               two components are mixed, each 250 mls and double-strength -
                        ·  Add the gelling agent (5 g agar or 2.5 g agarose) to 250 ml MQ
                        water in a 500 ml Schott bottle with a magnetic stirrer.
                        ·  Add 12.5 mls of filter-sterile x40 concentrate to 250 ml sterile MQ
                        water in a 250 ml Schott bottle. (full strength = 25ml/L in liquid media)
                        ·  Autoclave gelling agent, and double-strength nutrients.
                        ·  Final steps detailed below

 

· To prepare 500 ml of saline solid MLA medium:

three components are mixed, each 167 mls and triple-strength -

·  Add the gelling agent (5 g agar or 2.5 g agarose) to 167 ml MQ water in a 500 ml Schott bottle with a magnetic stirrer.

·  Add 12.5 mls of filter-sterile x40 concentrate to 167 mls MQ water.

·  Autoclave the gelling agent, the triple-strength nutrients and 167 mls of triple strength saline  

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Final steps in preparation of solid media:

·          Cool to 45 ⁰C in a water bath, and in the same bath, warm the CaCl₂ and NaHCO₃ and if appropriate Na₂SO₃.

·          On a magnetic stirrer plate in the laminar flow, place the bottle containing the gel. Stir gently while aseptically adding nutrients (and for saline media, adding seawater also).

·          Aseptically add 1 ml each of vitamins and CaCl₂ and if appropriate Na₂SO₃, and 10 ml of NaHCO₃.

·          Pour media into sterile petri plates or McCartney bottles***.

·          Dry agar/agarose plates in the laminar flow for 20 minutes prior to storing; and/or place McCartneys in a basket on a sharp angle until slopes are solid.

 

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***If using a dispenser, and in case problems arise during dispensing the solid media, it is advised to keep some hot sterile water for rinsing the dispenser aseptically.

 

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Mineral Medium II

Reference: Hughes, E. O., Gorham, P. R. and Zehnder, A. (1958) Canad. J. Microbiol. 4: 225-236.

Freshwater Algae

 

Stock Solutions	Per Liter distilled water  (dH2O)
1. NaNO3	1500.0 g
2. K2HPO4	370.0 g
3. MgSO4.7H2O	80.0 g
4. CaCl2.2H2O	40.0 g
5. Na2CO3	20.0 g
6. Na2SiO3.9H2O	60.0 g
7. Fe citrate:
Ferric citrate	6.0 g
Citric acid	6.0 g (autoclave to dissolve)
8. EDTA	1.0 g
9. PII Metal Mix	g / L dH2O	Add the Na2EDTA to ~750mL of dH2O in a volumetric flask and stir over low heat to dissolve.  Add each of the other constituents separately to ~200mL of dH2O and fully dissolve between additions.  Then add this second solution to the Na2EDTA and make up to 1L
Na2EDTA	6.0 g
FeCl3.6H2O	0.29 g
H3BO3	6.85 g
MnCl2.4H2O	0.86 g
ZnCl2	0.06 g
CoCl2.6H2O	0.026 g
Adjust pH to 7.8 - 8.0 with NaOH

 

Store all stock solutions in the refrigerator.

 

To Prepare Mineral Medium II

To 1 litre distilled water

Add 1mL of each stock solution (1 – 8).

Add 0.5mL PII Metal Mix stock solution (9)

Autoclave at 121°C (15PSI, 15 mins).

 

Mineral Medium II CSIRO working modification

This recipe closely resembles the Mineral Medium II listed previously. However it is made up using stocks from other media used in CMAR.

 

Stock Solution                        Source                         Add V

1. NaNO3                                                      D stock solution          15.0 mL

2. K2HPO4                                                    G stock solution          10.0 mL

3. MgSO4.7H2O                       MMII stock solution    1.0 mL

4. CaCl2.2H2O                         D stock solution          0.8 mL

5. Na2CO3                                                     MMII stock solution    1.0 mL

6. Na2SiO3.5H2O                     f stock solution            12.0 mL

7. Fe citrate:                            f stock solution            0.66 mL

8. Na2EDTA                            fE stock solution          0.14 mL

9. PII Metal Mix                      G stock solution          0.5 mL

Store all stock solutions in the refrigerator.

 

To Prepare Mineral Medium II

Add each stock solution (1 – 9) to 950 mL distilled water.

Autoclave at 121°C (15PSI, 15 mins).

 

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Porphyridium Medium

Medium for the heterotrophic dinoflagellate Crypthecodinium cohnii.

Reference: Starr, R. C. and Zeikus, J. A. (1993). UTEX - The Culture Collection of Algae at the University of Texas at Austin. J. Phycol. 29 (2) p94. (E.G. Pringsheim, pers. comm.)

To prepare medium:

For each 500ml of medium required combine:

distilled water               200 ml

flitered seawater             250 ml

yeast extract             0.5 g

tryptone                         0.5 g

autoclace to sterilise

add aseptically                    50 ml sterile soil extract.

Optional ingredients: agar at 7.5 g per litre to solidify.